The proposed research program will be a continuation of the development and utilization of magnetic resonance techniques to investigate the interactions of substrates with enzymes which catalyze reactions of nucleotides. The objective is to determine structures and conformations of enzyme--substrate complexes which resemble the transition state of the active enzyme system. Studies will focus initially on the enzymes creatine kinase and adenylate kinase from muscle tissues. These enzymes are pivotal components of systems for regulation and delivery of ATP to contractile proteins. Other glycolytic enzymes such as pyruvate kinase and hexokinase which are important in energy metabolism will also be examined. NMR and EPR spectroscopies are unique in their capacity to detect local conformational changes at the active site. With paramagnetic probes such as the metal ion activator, Mn(II), it is possible to determine distances between atoms at the active site, i.e. between Mn(II) and protons or phosphorus. EPR spectroscopy is particularly useful in detecting multiple conformational states of enzyme--substrate complexes. Identification of groups on the enzyme responsible for binding will be investigated by cross relaxation effects, i.e. nuclear Overhauser effects. Identification of the site of binding of appropriate diamagnetic metal ions will be investigated by hyperfine coupling to p31 of enzyme-bound substrates.